GeneRead Library Prep Kits provide an optimized and efficient workflow for library preparation in next-generation sequencing (NGS) applications. The kits allow fast and efficient preparation of DNA libraries that are for use on NGS platforms from Illumina and the Ion Torrent instrument from Life Technologies.

Genomic DNA (1 μg) from E. Coli strain DH10B was sheared using a Covaris instrument and made into an Illumina–compatible DNA library using the GeneRead Library Prep (I) Kit or a kit from another supplier. The library was eluted in 18 μl Buffer EB and library concentrations were determined by quantitative PCR.|A mixture of highly GC-rich Bordetella pertussis gDNA (GC content 67.7%) and highly AT-rich Streptobacillus moniliformis gDNA (GC content 26.3%) was pooled, made into an Illumina-compatible DNA library using the GeneRead Library (I) Core Kit, and amplified with the GeneRead Library (I) Amp Kit, which contains GeneRead HiFi Polymerase, or a kit from Supplier K. The amplified libraries were sequenced using the Illumina MiSeq instrument, and fidelity and sequence coverage were analyzed using the Galaxy platform. Low error rates and greater cumulated sequence coverage demonstrate that the GeneRead Library (I) Amp Kit provides superior library amplification compared to the kit from Supplier K.|The GeneRead Size Selection Kit effectively removes adapter dimers and adapter monomers following library preparation. A scaled-up image of the above data showing the correct size distribution of Illumina-compatible library fragments following size selection is shown in inset. FU: Fluorescence units.|A mixture of highly GC-rich Bordetella pertussis gDNA (GC content 67.7%) and highly AT-rich Streptobacillus moniliformis gDNA (GC content 26.3%) was pooled, made into an Illumina-compatible DNA library using the GeneRead Library Prep (I) Kit, and amplified with the GeneRead Library Amp (I) Kit, which contains GeneRead HiFi Polymerase, or a kit from Supplier K. The amplified libraries were sequenced using the Illumina MiSeq instrument, and fidelity and sequence coverage was analyzed using the Galaxy platform. The GeneRead Library Prep Kit provided greater sequence coverage in GC- and AT-rich areas of DNA, compared to the kit from Supplier K.|[A] Genomic DNA (50 ng) was sheared using a Covaris instrument and made into an Illumina-compatible DNA library using the GeneRead Library Prep (I) Kit. Sequencing using an Illumina MiSeq instrument revealed a median coverage of 49-fold with uniform coverage distribution. [B] Genomic DNA (1 μg) from E. coli strain DH10B was sheared and used to generate an Ion Torrent-compatible DNA library using the GeneRead Library Prep (L) Kit or a kit from another supplier. After amplification for 10 cycles, both libraries were sequenced on an Ion Torrent PGM instrument and the cumulated normalized coverage was analyzed.|Library Prep (I) Kit uses an optimized one-tube protocol, with fewer cleanup steps and optional high-fidelity library amplification. The hands-on time and total time required for preparation of library DNA is significantly reduced compared to the library preparation system from Supplier I.|The GeneRead Library Prep (L) Kit uses an optimized, one-tube procedure, with fewer cleanup steps and optional high-fidelity library amplification. The hands-on time and total time required for preparation of library DNA is significantly reduced compared to the library preparation system from Supplier L.|

Lower sequence bias when amplifying GC- and AT-rich regions

In standard PCR amplification procedures, regions of DNA with high AT or GC content can result in little or no amplification, leading to misleading sequence data and NGS results. The GeneRead HiFi Polymerase, together with its unique buffer formulation, ensures uniform amplification of genomic regions that contain highly variable GC content, thereby ensuring even coverage in subsequent sequencing reactions (see figure Better sequence coverage).

Principle

GeneRead Library Prep Kits use an optimized, one-tube workflow to reproducibly generate high yields of DNA library for use on NGS platforms from Illumina or the Ion Torrent instrument from Life Technologies. The fast, one-tube procedure uses fewer cleanup steps than library preparation workflows from other suppliers and includes an optional, high-fidelity library amplification step. GeneRead Library Prep Kits save time and effort while minimizing the variability caused by handing, as well as the risk of contamination (see figures Optimized workflow for Illumina-compatible DNA libraries and Optimized workflow for Ion Torrent-compatible DNA libraries).

Procedure

Procedure for generating Illumina-compatible DNA libraries

Samples consisting of longer DNA fragments are first sheared into a random library of fragments that are a median fragment size of 300 bp (when using the Illumina MiSeq instrument, version 1), or 500 bp (when using the Illumina MiSeq instrument, version 2), in length. Following fragmentation, the ends of the DNA fragments are repaired and an A-overhang is added at the 3'-end of each strand. Afterwards, adaptors, which are necessary for amplification and sequencing, are ligated to both ends of the DNA fragments. Barcode adapters, which contain a unique identifying sequence, are also available with the GeneRead Library Prep (I) Kit and enable multiplex sequencing reactions to be performed. Fragments are then size selected and purified. Cleanup and size selection can be done using the GeneRead Size Selection Kit (cat. no. 180514), which allows precise, column-based size selection of DNA (see figure Precise size selection). To ensure maximum yields from minimum amounts of starting material, an optional high-fidelity amplification step can also be performed.

Procedure for generating Ion Torrent-compatible DNA libraries

Samples consisting of longer DNA fragments are first sheared into a random library of fragments that are a median fragment size of 400 bp (when using the Ion Torrent PGM instrument), or 200 bp (when using the Ion Proton instrument), in length. Following fragmentation, the ends of the DNA fragments are repaired. Afterwards, adaptors, which are necessary for amplification and sequencing, are ligated to both ends of the DNA fragments. Barcode adapters, which contain a unique identifying sequence, are also available with the GeneRead Library Prep (L) Kit and enable multiplex sequencing reactions to be performed. Fragments are then size selected and purified. To ensure maximum yields from minimum amounts of starting material, an optional high-fidelity amplification step can be performed.

Applications

GeneRead Library Prep Kits allow fast and efficient preparation of DNA libraries that are optimized for use on NGS platforms from Illumina and the Ion Torrent instrument from Life Technologies.